Objective:To explore the possible effects and mechanism of Zhizhu Decoction(ZZD)on the pathophysiology of slow transit constipation(STC).Methods A total of 54 C57BL/6 mice was randomly divided into the following 6 gro...Objective:To explore the possible effects and mechanism of Zhizhu Decoction(ZZD)on the pathophysiology of slow transit constipation(STC).Methods A total of 54 C57BL/6 mice was randomly divided into the following 6 groups by a random number table,including control,STC model(model),positive control,and low-,medium-and high-doses ZZD treatment groups(5,10,20 g/kg,namely L,M-,and H-ZZD,respectively),9 mice in each group.Following 2-week treatment,intestinal transport rate(ITR)and fecal water content were determined,and blood and colon tissue samples were collected.Hematoxylin-eosin and periodic acid-Schiff staining were performed to evaluate the morphology of colon tissues and calculate the number of goblet cells.To determine intestinal permeability,serum levels of lipopolysaccharide(LPS),low-density lipoprotein(LDL)and mannose were measured using enzyme-linked immunosorbent assay(ELISA).Western blot analysis was carried out to detect the expression levels of intestinal tight junction proteins zona-occludens-1(ZO-1),claudin-1,occludin and recombinant mucin 2(MUC2).The mRNA expression levels of inflammatory cytokines including tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,IL-4,IL-10 and IL-22 were determined using reverse transcription-quantitative reverse transcription reaction.Colon indexes of oxidative stress were measured by ELISA,and protein expression levels of colon silent information regulator 1/forkhead box O transcription factor 1(SIRT1/FoxO1)antioxidant signaling pathway were detected by Western blot.Results Compared with the model group,ITR and fecal moisture were significantly enhanced in STC mice in the M-ZZD and H-ZZD groups(P<0.01).Additionally,ZZD treatment notably increased the thickness of mucosal and muscular tissue,elevated the number of goblet cells in the colon of STC mice,reduced the secretion levels of LPS,LDL and mannose,and upregulated ZO-1,claudin-1,occludin and MUC2 expressions in the colon in a dose-dependent manner,compared with the model group(P<0.05 or P<0.01).In addition,ZZD 展开更多
DNA结合抑制蛋白-1(inhibitor of DNA binding 1,ID1)在结肠癌、肺癌和乳腺癌等多种肿瘤组织中异常高表达,并与肿瘤恶性进展及患者不良预后密切相关。已有研究发现,ID1在结肠癌中可以维持肿瘤细胞干细胞样特性,介导肿瘤干细胞耐药,但其...DNA结合抑制蛋白-1(inhibitor of DNA binding 1,ID1)在结肠癌、肺癌和乳腺癌等多种肿瘤组织中异常高表达,并与肿瘤恶性进展及患者不良预后密切相关。已有研究发现,ID1在结肠癌中可以维持肿瘤细胞干细胞样特性,介导肿瘤干细胞耐药,但其分子生物学机制并未被完全阐明。通过免疫印迹、基因集富集分析、双荧光素酶报告基因检测及实时定量PCR,本研究发现ID1可从转录水平上调维持肿瘤细胞干性的关键转录因子八聚体结合蛋白(octamer binding transcription factor,OCT4)的蛋白表达及信号通路活性。利用体外成球实验,本研究证明过表达OCT4可逆转敲低ID1对干细胞成球能力的抑制作用。在进一步的机制研究中,利用JASPAR及GEPIA数据库,本研究预测并得到能够负向调控OCT4转录的转录抑制因子叉头蛋白D3(forkhead box D3,FOXD3)。通过免疫共沉淀、免疫荧光共聚焦及实时定量PCR等方法,本研究最终证明ID1通过与转录抑制因子FOXD3相互作用,抑制其活性从而上调OCT4的转录及信号通路活性。综上,本研究为结肠癌干细胞的分子调控机制提供了新的理论基础,研究中发现的ID1-FOXD3蛋白-蛋白相互作用为结肠癌治疗提供了新的潜在干预靶点。展开更多
基金Supported by the General Project of National Natural Science Foundation of China(No.82074429)。
文摘Objective:To explore the possible effects and mechanism of Zhizhu Decoction(ZZD)on the pathophysiology of slow transit constipation(STC).Methods A total of 54 C57BL/6 mice was randomly divided into the following 6 groups by a random number table,including control,STC model(model),positive control,and low-,medium-and high-doses ZZD treatment groups(5,10,20 g/kg,namely L,M-,and H-ZZD,respectively),9 mice in each group.Following 2-week treatment,intestinal transport rate(ITR)and fecal water content were determined,and blood and colon tissue samples were collected.Hematoxylin-eosin and periodic acid-Schiff staining were performed to evaluate the morphology of colon tissues and calculate the number of goblet cells.To determine intestinal permeability,serum levels of lipopolysaccharide(LPS),low-density lipoprotein(LDL)and mannose were measured using enzyme-linked immunosorbent assay(ELISA).Western blot analysis was carried out to detect the expression levels of intestinal tight junction proteins zona-occludens-1(ZO-1),claudin-1,occludin and recombinant mucin 2(MUC2).The mRNA expression levels of inflammatory cytokines including tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,IL-4,IL-10 and IL-22 were determined using reverse transcription-quantitative reverse transcription reaction.Colon indexes of oxidative stress were measured by ELISA,and protein expression levels of colon silent information regulator 1/forkhead box O transcription factor 1(SIRT1/FoxO1)antioxidant signaling pathway were detected by Western blot.Results Compared with the model group,ITR and fecal moisture were significantly enhanced in STC mice in the M-ZZD and H-ZZD groups(P<0.01).Additionally,ZZD treatment notably increased the thickness of mucosal and muscular tissue,elevated the number of goblet cells in the colon of STC mice,reduced the secretion levels of LPS,LDL and mannose,and upregulated ZO-1,claudin-1,occludin and MUC2 expressions in the colon in a dose-dependent manner,compared with the model group(P<0.05 or P<0.01).In addition,ZZD
文摘DNA结合抑制蛋白-1(inhibitor of DNA binding 1,ID1)在结肠癌、肺癌和乳腺癌等多种肿瘤组织中异常高表达,并与肿瘤恶性进展及患者不良预后密切相关。已有研究发现,ID1在结肠癌中可以维持肿瘤细胞干细胞样特性,介导肿瘤干细胞耐药,但其分子生物学机制并未被完全阐明。通过免疫印迹、基因集富集分析、双荧光素酶报告基因检测及实时定量PCR,本研究发现ID1可从转录水平上调维持肿瘤细胞干性的关键转录因子八聚体结合蛋白(octamer binding transcription factor,OCT4)的蛋白表达及信号通路活性。利用体外成球实验,本研究证明过表达OCT4可逆转敲低ID1对干细胞成球能力的抑制作用。在进一步的机制研究中,利用JASPAR及GEPIA数据库,本研究预测并得到能够负向调控OCT4转录的转录抑制因子叉头蛋白D3(forkhead box D3,FOXD3)。通过免疫共沉淀、免疫荧光共聚焦及实时定量PCR等方法,本研究最终证明ID1通过与转录抑制因子FOXD3相互作用,抑制其活性从而上调OCT4的转录及信号通路活性。综上,本研究为结肠癌干细胞的分子调控机制提供了新的理论基础,研究中发现的ID1-FOXD3蛋白-蛋白相互作用为结肠癌治疗提供了新的潜在干预靶点。
